RAVA is derived from LAVA, but for nonlongitudinal sequence data. RAVA takes FASTQ files (for every sample in your analysis), a metadata sheet (providing info on what day or passage each sample was collected), and a reference genome either by accession number or your own FASTA and GFF. Output will be displayed as an interactive graph in your web browser, and a table of all mutations of all samples.
Fastqs should be trimmed before running through the pipeline. You can use single-end reads (default) or paired-end reads (interleaved format) as input.
- Install nextflow.
- Make sure you move nextflow to a directory in your PATH variable.
- Install docker.
- If running on the cloud setup nextflow tower
The input metadata file has only two inputs required
Sample: Name of fastq and path if not in current directory
Passage: Sample name to be displayed on html
| Sample | Passage |
|---|---|
| Example_fastq/SRR24099553.fastq | SRR24099553 |
| Example_fastq/SRR24099554.fastq | SRR24099554 |
RAVA accepts reference input as .gff, .gb or .gbk (Genbank Annotation) or Genbank accession
If GFF or GBK is used as input they are both passed into --GFF and require a fasta input --FASTA
Important
It is strongly recommended to use GBK as your input as is easiest to format
| File | Notes |
|---|---|
| .gff | If GFF is chosen as input please see the GFF reformating guide as a standard GFF file is not accepted as input. A fasta file is required for input. |
| .gb or .gbk | Use --GFF as input for your .gb file, a fasta file is required through --FASTA. A genbank file can be downloaded from NCBI or exported from Geneious. If exporting from Geneious click on an annotated fasta and export as .gbk file |
Warning
Genbank files may need to be reformated as CDS is the only input accepted in the pipeline for the protein to be recognized. See reformating Genbank file see Reformating Genbank File.
Warning
The pipeline may crash on the standard nextflow config, it is recommended to use the cloud or MORE for local runs when running more computationally intensive processes.
| Command | Description |
|---|---|
--METADATA |
Input metadata CSV |
--OUTDIR |
Output directory |
--FASTA |
Specify a reference fasta to map samples to. This must be the same fasta as your annotation file (.gb or .gff). This option must be used with the --GFF flag to specify the protein annotations relative to the start of this fasta. |
--GFF |
Specify a reference gff, gb or gbk file with the protein annotations for the reference fasta supplied with the --FASTA flag. This option must be paired with the --FASTA flag. |
--NAME |
Optional flag, Change name of output html. |
--ALLELE_FREQ |
Optional flag, Specify an allele frequency percentage to cut off - with a minimum of 1 percent - in whole numbers. |
--PAIRING |
Optional flag for paired-end interleaved input. |
--DEDUPLICATE |
Optional flag, will perform automatic removal of PCR duplicates via DeDup. |
-resume |
nextflow will pick up where it left off if the previous command was interrupted for some reason. |
-with-docker ubuntu:18.04 |
Runs command with Ubuntu docker. |
-with-trace |
Outputs a trace.txt that shows which processes end up in which work/ folders. |
-profile |
standard: For less computationally intensive systems run locally, not reccommended |
MORE: For running on the more computationally strong local systems, used for lab i9 imacs |
|
Cloud: For running on the cloud adds more computational power |
|
-c |
Add you nextflow config file to access cloud |
-with-tower |
Monitor your run with nextflow tower |
Run locally with standard profile with .gb file
nextflow run greninger-lab/RAVA_Pipeline -r main\
--OUTDIR example_output/ \
--GFF Examples/Example.gb \
--FASTA Examples/Example.fasta \
--METADATA Examples/Example_metadata.csv \
-with-docker ubuntu:18.04 \
-profile standard
Run on cloud with .gff file
nextflow run greninger-lab/RAVA_Pipeline -r main \
--OUTDIR example_output/ \
--GFF Examples/Example.gff \
--FASTA Examples/Example.fasta \
--METADATA Examples/Example_metadata.csv \
-with-docker ubuntu:18.04 \
-profile Cloud \
-c your_nextflow_aws.config \
-with-tower
Run locally with more computational power
nextflow run greninger-lab/RAVA_Pipeline -r main\
--OUTDIR example_output/ \
--GFF Examples/Example.gb \
--FASTA Examples/Example.fasta \
--METADATA Examples/Example_metadata.csv \
-with-docker ubuntu:18.04 \
-profile More
flowchart TD;
CreateGFF-->Align_samples;
Align_samples-->Pipeline_prep;
Pipeline_prep-->Create_VCF;
Create_VCF-->Extract_variants;
Extract_variants-->Annotate_complex;
Annotate_complex-->Generate_output;
Install sratoolkit
On Mac you can use brew:
brew install sratoolkitMake an Example_fastq folder
mkdir Example_fastqEnter Example_fastq folder
cd Example_fastqDownload samples from SRA and place them in the Example_fastq folder.
fasterq-dump --split-files SRR24099553
fasterq-dump --split-files SRR24099554 If you use paired-end, combine R1 and R2 reads in an interleaved format (remember to use flag '--PAIRING' when running RAVA), for example, using seqtk:
for i in *_1.fastq
do
base=$(basename $i _1.fastq)
seqtk mergepe "${base}_1.fastq" "${base}_2.fastq" > "${base}.fastq"
doneRemove unpaired reads
rm *_1.fastq
rm *_2.fastqExit folder
cd ..Run example workflow or use one from above
With gb file
nextflow run greninger-lab/RAVA_Pipeline -r main --OUTDIR example_output/ --GFF Examples/Example.gb --FASTA Examples/Example.fasta --METADATA Examples/Example_metadata.csv -with-docker ubuntu:18.04 -profile standard
With gff file
nextflow run greninger-lab/RAVA_Pipeline -r main --OUTDIR example_output/ --GFF Examples/Example.gff --FASTA Examples/Example.fasta --METADATA Examples/Example_metadata.csv -with-docker ubuntu:18.04 -profile standard
example_output
├── RAVA_plots.html # Rava html visualization file open in browser
├── all_files
│ ├── mat_peptides_additions.txt # If mature peptides are included in annotation they are listed here
│ └── ribosomal_start.txt # First peptide
├── bam_files
│ ├── SRR24099553.fastq.bam # bam file from alignment to reference file
│ └── SRR24099554.fastq.bam
├── complex.log # Complex annotations are listed here if there are any
├── genomecov
│ ├── SRR24099553.fastq.genomecov # genome coverage file for sample
│ └── SRR24099554.fastq.genomecov
├── reads.csv # reads stats file
├── vcf_files
│ ├── SRR24099553.fastq.vcf # vcf for sample
│ └── SRR24099554.fastq.vcf
└── visualization.csv # csv used to generate visualization, shows all annotated mutations
Sometime a virus will have a large polyprotein. For a genbank file this will be labeled as a CDS and all other proteins will be labeled as mat_peptide.
This will produce an output visualization where the polyprotein is the only ORF annotated and mature peptides are green arrows.
It is recommended to rename the large polyprotein to anything other than CDS and rename the mature peptides to CDS for them to display with labels.
From
LOCUS AB016162 15894 bp RNA linear VRL 21-SEP-2000
DEFINITION Measles virus genomic RNA, complete sequence.
ACCESSION AB016162
VERSION AB016162.1
KEYWORDS nonstructural C protein; L; large polymerase; H; hemagglutinin
protein; F; fusion protein; M; matrix protein; C; P;
phosphoprotein; N; nucleocapsid protein.
SOURCE Measles morbillivirus
ORGANISM Measles morbillivirus
Viruses; Riboviria; Orthornavirae; Negarnaviricota;
Haploviricotina; Monjiviricetes; Mononegavirales; Paramyxoviridae;
Orthoparamyxovirinae; Morbillivirus; Morbillivirus hominis.
REFERENCE 1
AUTHORS Takeuchi,K., Miyajima,N., Kobune,F. and Tashiro,M.
TITLE Comparative nucleotide sequence analyses of the entire genomes of
B95a cell-isolated and vero cell-isolated measles viruses from the
same patient
JOURNAL Virus Genes 20 (3), 253-257 (2000)
PUBMED 10949953
REFERENCE 2 (bases 1 to 15894)
AUTHORS Takeuchi,K., Tanabayashi,K. and Tashiro,M.
TITLE Direct Submission
JOURNAL Submitted (10-JUL-1998) Kaoru Takeuchi, National Institute of
Infectious Diseases, Viral Disease and Vaccine Contorol; 4-7-1
Gakuen, Musashi-murayama, Tokyo 208-0011, Japan
(E-mail:ktake@nih.go.jp, Tel:81-42-561-0771(ex.530),
Fax:81-42-567-5631)
COMMENT On Dec 14, 1998 this sequence version replaced gi:3986256.
Sequence updated (21-Jul-1998)
Sequence updated (11-Dec-1998).
FEATURES Location/Qualifiers
CDS 1..15894
/organism="Measles morbillivirus"
/mol_type="genomic RNA"
/strain="Ichinose-B95a"
/db_xref="taxon:11234"
gene 108..1685
/gene="N"
mat_peptide 108..1685
/gene="N"
/codon_start=1
/product="nucleocapsid protein"
/protein_id="BAA34977.1"
/translation="MATLLRSLALFKRNKDKPPITSGSGGAIRGIKHIIIVPIPGDSS
ITTRSRLLDRLVRLIGNPDVSGPKLTGALIGILSLFVESPGQLIQRITDDPDVSIRLL
EVVQSDQSQSGLTFASRGTNMEDEADQYFSHDDPSSSDQSRSGWFENKEISDIEVQDP
EGFNMILGTILAQIWVLLAKAVTAPDTAADSELRRWIKYTQQRRVVGEFRLERKWLDV
VRNRIAEDLSLRRFMVALILDIKRTPGNKPRIAEMICDIDTYIVEAGLASFILTIKFG
IETMYPALGLHEFAGELSTLESLMNLYQQMGETAPYMVILENSIQNKFSAGSYPLLWS
YAMGVGVELENSMGGLNFGRSYFDPAYFRLGQEMVRRSAGKVSSTLASELGITAEDAR
LVSEIAMHTTEDRISRAVGPRQAQVSFLHGDQSENELPGLGGKEDRRVKQGRGEARES
YRETGSSRASDARAAHPPTSMPLDIDTASESGQDPQDSRRSADALLRLQAMAGILEEQ
GSDTDTPRVYNDRDLLD"
gene 1807..3330
/gene="P"
mat_peptide 1807..3330
/gene="P"
/note="additional G insertion at 2499:V mRNA"
/codon_start=1
/product="phosphoprotein"
/protein_id="BAA34978.1"
/translation="MAEEQARHVKNGLECIRALKAEPIGSLAVEEAMAAWSEISDNPG
QDRATCKEEEAGSSGLSKPCLSAIGSTEGGAPRIRGQGSGESDDDAETLGIPSRNLQA
SSTGLQCYHVYDHSGEAVKGIQDADSIMVQSGLDGDSTLSGGDDESENSDVDIGEPDT
EGYAITDRGSAPISMGFRASDVETAEGGEIHELLKLQSRGNNFPKLGKTLNVPPPPNP
SRASTSETPIKKGTDARLASFGTEIASLLTGGATQCARKSPSEPSGPGAPAGNVPECV
SNAALIQEWTPESGTTISPRSQNNEEGGDYYDDELFSDVQDIKTALAKIHEDNQKIIS
KLESLLLLKGEVESIKKQINRQNISISTLEGHLSSIMIAIPGLGKDPNDPTADVELNP
DLKPIIGRDSGRALAEVLKKPVASRQLQGMTNGRTSSRGQLLKEFQLKPIGKKVSSAV
GFVPDTGPASRSVIRSIIKSSRLEEDRKRYLMTLLDDIKGANDLAKFHQMLMKIIMK"
mat_peptide 1829..2389
/gene="P"
/codon_start=1
/product="nonstructural C protein"
/protein_id="BAA34979.1"
/translation="MSKTDWNASGLSRPSPSAHWPSRKPWQHGQKYQTTQDRTEPPAR
KRRQAVRVSANHASQQLDQLKAVHLASAVRDLEKAMTTLKLWESPQEISRHQALGYSV
IMFMITAVKRLRESKMLTLSWFNQALMVIAPSQEETMNLKTAMWILANLIPRDMLSLT
GDLLPSLWGSGLLMLKLQKEGRSTSS"
gene 3438..4445
/gene="M"
mat_peptide 3438..4445
/gene="M"
/codon_start=1
/product="matrix protein"
/protein_id="BAA34980.1"
/translation="MTEIYDFDKSAWDIKGSIAPIQPTTYSDGRLVPQVRVIDPGLGD
RKDECFMYMFLLGVVEDSDPLGPPIGRAFGSLPLGVGRSTAKPEELLKEATELDIVVR
RTAGLNEKLVFYNNTPLTLLTPWRKVLTTGSVFNANQVCNAVNLIPLDTPQRFRVVYM
SITRLSDNGYYTVPRRMLEFRSVNAVAFNLLVTLRIDKAIGPGKIIDNAEQLPEATFM
VHIGNFRRKKSEVYSADYCKMKIEKMGLVFALGGIGGTSLHIRSTGKMSKTLHAQLGF
KKTLCYPLMDINEDLNRLLWRSRCKIVRIQAVLQPSVPQEFRIYDDVIINDDQGLFKV
L"
gene 5458..7110
/gene="F"
mat_peptide 5458..7110
/gene="F"
/note="additional G inserted at 2499:V"
/codon_start=1
/product="fusion protein"
/protein_id="BAA34981.1"
/translation="MGLKVNVSAIFMAVLLTLQTPTGQIHWGNLSKIGVVGIGSASYK
VMTRSSHQSLVIKLMPNITLLNNCTRVEIAEYRRLLRTVLEPIRDALNAMTQNIRPVQ
SVASSRRHKRFAGVVLAGAALGVATAAQITAGIALHQSMLNSQAIDNLRASLETTNQA
IEAIRQAGQEMILAVQGVQDYINNELIPSMNQLSCDLIGQKLGLKLLRYYTEILSLFG
PSLRDPISAEISIQALSYALGGDINKVLEKLGYSGGDLLGILESRGIKARITHVDTES
YFIVLSIAYPTLSEIKGVIVHRLEGVSYNIGSQEWYTTVPKYVATQGYLISNFDESSC
TFMPEGTVCSQNALYPMSPLLQECLRGSTKSCARTLVSGSFGNRFILSQGNLIANCAS
ILCKCYTTGTIINQDPDKILTYIAADHCPVVEVNGVTIQVGSRRYPDAVYLHRIDLGP
PISLERLDVGTNLGNAIAKLEDAKELLESSDQILRSMKGLSSTSIVYILIAVCLGGLI
GIPALICCCRGRCNKKGEQVGMSRPGLKPDLTGTSKSYVRSL"
To
LOCUS AB016162 15894 bp RNA linear VRL 21-SEP-2000
DEFINITION Measles virus genomic RNA, complete sequence.
ACCESSION AB016162
VERSION AB016162.1
KEYWORDS nonstructural C protein; L; large polymerase; H; hemagglutinin
protein; F; fusion protein; M; matrix protein; C; P;
phosphoprotein; N; nucleocapsid protein.
SOURCE Measles morbillivirus
ORGANISM Measles morbillivirus
Viruses; Riboviria; Orthornavirae; Negarnaviricota;
Haploviricotina; Monjiviricetes; Mononegavirales; Paramyxoviridae;
Orthoparamyxovirinae; Morbillivirus; Morbillivirus hominis.
REFERENCE 1
AUTHORS Takeuchi,K., Miyajima,N., Kobune,F. and Tashiro,M.
TITLE Comparative nucleotide sequence analyses of the entire genomes of
B95a cell-isolated and vero cell-isolated measles viruses from the
same patient
JOURNAL Virus Genes 20 (3), 253-257 (2000)
PUBMED 10949953
REFERENCE 2 (bases 1 to 15894)
AUTHORS Takeuchi,K., Tanabayashi,K. and Tashiro,M.
TITLE Direct Submission
JOURNAL Submitted (10-JUL-1998) Kaoru Takeuchi, National Institute of
Infectious Diseases, Viral Disease and Vaccine Contorol; 4-7-1
Gakuen, Musashi-murayama, Tokyo 208-0011, Japan
(E-mail:ktake@nih.go.jp, Tel:81-42-561-0771(ex.530),
Fax:81-42-567-5631)
COMMENT On Dec 14, 1998 this sequence version replaced gi:3986256.
Sequence updated (21-Jul-1998)
Sequence updated (11-Dec-1998).
FEATURES Location/Qualifiers
source 1..15894
/organism="Measles morbillivirus"
/mol_type="genomic RNA"
/strain="Ichinose-B95a"
/db_xref="taxon:11234"
gene 108..1685
/gene="N"
CDS 108..1685
/gene="N"
/codon_start=1
/product="nucleocapsid protein"
/protein_id="BAA34977.1"
/translation="MATLLRSLALFKRNKDKPPITSGSGGAIRGIKHIIIVPIPGDSS
ITTRSRLLDRLVRLIGNPDVSGPKLTGALIGILSLFVESPGQLIQRITDDPDVSIRLL
EVVQSDQSQSGLTFASRGTNMEDEADQYFSHDDPSSSDQSRSGWFENKEISDIEVQDP
EGFNMILGTILAQIWVLLAKAVTAPDTAADSELRRWIKYTQQRRVVGEFRLERKWLDV
VRNRIAEDLSLRRFMVALILDIKRTPGNKPRIAEMICDIDTYIVEAGLASFILTIKFG
IETMYPALGLHEFAGELSTLESLMNLYQQMGETAPYMVILENSIQNKFSAGSYPLLWS
YAMGVGVELENSMGGLNFGRSYFDPAYFRLGQEMVRRSAGKVSSTLASELGITAEDAR
LVSEIAMHTTEDRISRAVGPRQAQVSFLHGDQSENELPGLGGKEDRRVKQGRGEARES
YRETGSSRASDARAAHPPTSMPLDIDTASESGQDPQDSRRSADALLRLQAMAGILEEQ
GSDTDTPRVYNDRDLLD"
gene 1807..3330
/gene="P"
CDS 1807..3330
/gene="P"
/note="additional G insertion at 2499:V mRNA"
/codon_start=1
/product="phosphoprotein"
/protein_id="BAA34978.1"
/translation="MAEEQARHVKNGLECIRALKAEPIGSLAVEEAMAAWSEISDNPG
QDRATCKEEEAGSSGLSKPCLSAIGSTEGGAPRIRGQGSGESDDDAETLGIPSRNLQA
SSTGLQCYHVYDHSGEAVKGIQDADSIMVQSGLDGDSTLSGGDDESENSDVDIGEPDT
EGYAITDRGSAPISMGFRASDVETAEGGEIHELLKLQSRGNNFPKLGKTLNVPPPPNP
SRASTSETPIKKGTDARLASFGTEIASLLTGGATQCARKSPSEPSGPGAPAGNVPECV
SNAALIQEWTPESGTTISPRSQNNEEGGDYYDDELFSDVQDIKTALAKIHEDNQKIIS
KLESLLLLKGEVESIKKQINRQNISISTLEGHLSSIMIAIPGLGKDPNDPTADVELNP
DLKPIIGRDSGRALAEVLKKPVASRQLQGMTNGRTSSRGQLLKEFQLKPIGKKVSSAV
GFVPDTGPASRSVIRSIIKSSRLEEDRKRYLMTLLDDIKGANDLAKFHQMLMKIIMK"
CDS 1829..2389
/gene="P"
/codon_start=1
/product="nonstructural C protein"
/protein_id="BAA34979.1"
/translation="MSKTDWNASGLSRPSPSAHWPSRKPWQHGQKYQTTQDRTEPPAR
KRRQAVRVSANHASQQLDQLKAVHLASAVRDLEKAMTTLKLWESPQEISRHQALGYSV
IMFMITAVKRLRESKMLTLSWFNQALMVIAPSQEETMNLKTAMWILANLIPRDMLSLT
GDLLPSLWGSGLLMLKLQKEGRSTSS"
gene 3438..4445
/gene="M"
CDS 3438..4445
/gene="M"
/codon_start=1
/product="matrix protein"
/protein_id="BAA34980.1"
/translation="MTEIYDFDKSAWDIKGSIAPIQPTTYSDGRLVPQVRVIDPGLGD
RKDECFMYMFLLGVVEDSDPLGPPIGRAFGSLPLGVGRSTAKPEELLKEATELDIVVR
RTAGLNEKLVFYNNTPLTLLTPWRKVLTTGSVFNANQVCNAVNLIPLDTPQRFRVVYM
SITRLSDNGYYTVPRRMLEFRSVNAVAFNLLVTLRIDKAIGPGKIIDNAEQLPEATFM
VHIGNFRRKKSEVYSADYCKMKIEKMGLVFALGGIGGTSLHIRSTGKMSKTLHAQLGF
KKTLCYPLMDINEDLNRLLWRSRCKIVRIQAVLQPSVPQEFRIYDDVIINDDQGLFKV
L"
gene 5458..7110
/gene="F"
CDS 5458..7110
/gene="F"
/note="additional G inserted at 2499:V"
/codon_start=1
/product="fusion protein"
/protein_id="BAA34981.1"
/translation="MGLKVNVSAIFMAVLLTLQTPTGQIHWGNLSKIGVVGIGSASYK
VMTRSSHQSLVIKLMPNITLLNNCTRVEIAEYRRLLRTVLEPIRDALNAMTQNIRPVQ
SVASSRRHKRFAGVVLAGAALGVATAAQITAGIALHQSMLNSQAIDNLRASLETTNQA
IEAIRQAGQEMILAVQGVQDYINNELIPSMNQLSCDLIGQKLGLKLLRYYTEILSLFG
PSLRDPISAEISIQALSYALGGDINKVLEKLGYSGGDLLGILESRGIKARITHVDTES
YFIVLSIAYPTLSEIKGVIVHRLEGVSYNIGSQEWYTTVPKYVATQGYLISNFDESSC
TFMPEGTVCSQNALYPMSPLLQECLRGSTKSCARTLVSGSFGNRFILSQGNLIANCAS
ILCKCYTTGTIINQDPDKILTYIAADHCPVVEVNGVTIQVGSRRYPDAVYLHRIDLGP
PISLERLDVGTNLGNAIAKLEDAKELLESSDQILRSMKGLSSTSIVYILIAVCLGGLI
GIPALICCCRGRCNKKGEQVGMSRPGLKPDLTGTSKSYVRSL"
Perhaps the most difficult aspect of running this program is properly formatting your reference fasta and .gff files. In order to have a longitudinal analysis that makes sense, you need to specify a fasta file containing the majority consensus for the first sample. This allows you to examine minor variants in your first sample properly. If you use a fasta that is not representative of your first sample RAVA will Genbank many mutations at 100% allele frequency in your first sample.
Example: Using the Template GFF
Use your favorite method of generating a consensus fasta for your first set of reads (we mainly use Geneious). Once this is done you need to make your .gff file. However, ANNOVAR requires a VERY strict formatting of these gff files.
The easiest way of generating a new gff file is to edit gene/CDS/transcript names and locations in the provided Example1_ref.gff. We highly recommend this method to avoid lots of formatting issues.
To do this you first need to have your start and end nucleotide postitions for your protein locations - these must be relative to the start of the provided reference fasta. (So if you're using a reference fasta that you added 200 Ns to the start of the sequence, all protein starts and stops would need to be increased by 200).
Below is a visual guide of what you need to change the template Example1_ref.gff for your purposes.
The color coded regions are the only portions that need to be changed. (The first two lines of the .gff files are comments and can be safely ignored.)
Proteins are coded by 3 tab separated lines (gene, CDS, transcript).
- The first column must be the name of your .fasta reference sequence. (So if the first line of your reference .fasta is >example, the first column of each row should read
example. Here, the name isWSN_reference2. - The fourth column should contain your start and end nucleotide positions of protein locations. Change the numbers to match your fasta file. Make sure to keep the blocks of 3, so that there are correct protein locations for each of gene, CDS, transcript.
- The last column has protein names that need to be replaced. Make sure you are replacing after both the
ID=and theParent=.
Specific Requirements
If you don't want to use the template GFF, or want to troubleshoot any problems with the GFF that may be popping up, here are the specific formatting requirements for each column.
| Fasta Name | Source | Feature | Start | End | Score | Strand | Phase | Attributes |
|---|---|---|---|---|---|---|---|---|
| Must match the name of your .fasta reference sequence: both the first line, and the file name. | Anything in this column. | One of 3 things: gene, CDS, transcript. CDS must be in all caps. Each protein MUST have all 3 features. | Beginning position of the protein. | End position of the protein. | Only contains "." | Only contains "+". | Contains a "0" for all CDS lines, and "." for all others. | Contains ID=feature type, where feature type is one of gene, CDS, or transcript, followed by the protein name. For CDS lines, it must also contain a Parent=transcript: identifier, followed by the protein name. For transcript lines, it must also contain a Parent=gene: identifier, followed by the protein name. All lines must end with biotype=protein_coding. Each of these tags should be separated by semicolons. |
If you experience any difficulties doing this, or have any other questions about RAVA, feel free to email us at uwvirongs@gmail.com and we'll be happy to help you out!